Insulin resistance is a crucially important metabolic abnormality in diet-induced obesity and type 2 diabetes. Endothelial dysfunction exists in insulin-resistant states and may represent an important early event in the development of atherosclerosis. Reduced endothelium-derived nitric oxide (NO) contributes to endothelial dysfunction. The bioavailability of NO depends on a fine balance between NO production via endothelial NO synthase (eNOS) and inactivation of NO by reactive oxygen species such as superoxide anion (O2-). Endothelial production of NO requires the phosphorylation of specific eNOS residues by Akt/protein kinase B (Akt) and other upstream kinases, as well as the formation of eNOS homodimers. Based on our preliminary results and data from others, endothelial dysfunction that exists in mice with diet-induced obesity might be precipitated by impaired Akt-mediated eNOS phosphorylation in the vasculature and by the disruption of eNOS homodimers secondary to increased superoxide (O2-) production. We will test the overall hypothesis that accumulation of the sphingolipid ceramide contributes importantly to the pathogenesis of vascular dysfunction in insulin resistant states by reducing NO production via both of these mechanisms. In Aim 1 we will determine if increasing ceramide concentrations in bovine aortic endothelial cells (BAECs) impairs insulin signaling and decreases eNOS function. Two hypotheses will be tested: 1) Ceramide antagonizes insulin-mediated Akt and eNOS phosphorylation and stimulates MAPK signaling resulting in reduced NO production and increased endothelin-1 production by BAECs; and 2) Ceramide increases O2- to an extent that peroxynitrite formation disrupts eNOS homodimers and leads to reduced NO production by BAECs. In Aim 2 we will define the contribution from ceramide to impaired insulin-mediated signal transduction in the vasculature, reduced endothelium-dependent vasorelaxation, and systemic hypertension that we have observed in mice with diet- induced obesity. Two hypotheses will be tested in mice with diet-induced obesity: 1) Ceramide accumulation reduces NO bioavailability by impairing insulin-mediated Akt phosphorylation and eNOS phosphorylation in the vasculature; and 2) Ceramide accumulation reduces NO bioavailability by potentiating O2- -induced peroxynitrite formation which ultimately disrupts eNOS homodimer formation in the vasculature. Two strategies will be used to limit ceramide accumulation in mice with diet-induced obesity: pharmacological inhibition of the rate-limiting enzyme responsible for ceramide synthesis; and a novel knockout model wherein ceramide synthesis is prevented. Information generated by testing our hypotheses will provide important insight into understanding the contribution from ceramide to cardiovascular defects that exist in mice with diet-induced obesity. [unreadable] [unreadable] [unreadable] [unreadable]